Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof

ABSTRACT

One can impart outstanding resistance against collapse under pressure to gas-filled microvesicle used as contrast agents in ultrasonic echography by using as fillers gases whose solubility in water, expressed in liter of gas by liter of water under standard conditions, divided by the square root of the molecular weight does not exceed 0.003.

TECHNICAL FIELD

[0001] The present invention concerns stable dispersions or compositionsof gas filled microvesicles in aqueous carrier liquids. Thesedispersions are generally usable for most kinds of applicationsrequiring gases homogeneously dispersed in liquids. One notableapplication for such dispersions is to be injected into living beings,for instance for ultrasonic echography and other medical applications.The invention also concerns the methods for making the foregoingcompositions including some materials involved in the preparations, forinstance pressure-resistant gas-filled microbubbles, microcapsules andmicroballoons.

BACKGROUND OF INVENTION

[0002] It is well known that microbodies or microglobules of air or gas(defined here as microvesicles), e.g. microbubbles or microballoons,suspended in a liquid are exceptionally efficient ultrasound reflectorsfor echography. In this disclosure the term of “microbubble”specifically designates hollow spheres or globules, filled with air or agas, in suspension in a liquid which generally result from theintroduction therein of air or gas in divided form, the liquidpreferably also containing surfactants or tensides to control thesurface properties and the stability of the bubbles. The term of“microcapsule” or “microballoon” designates preferably air or gas-filledbodies with a material boundary or envelope. i.e. a polymer membranewall. Both microbubbles and microballoons are useful as ultrasoniccontrast agents. For instance injecting into the bloodstream of livingbodies suspensions of air-filled microbubbles or microballoons (in therange of 0.5 to 10 μm) in a carrier liquid will strongly reinforceultrasonic echography imaging, thus aiding in the visualization ofinternal organs. Imaging of vessels and internal organs can stronglyhelp in medical diagnosis, for instance for the detection ofcardiovascular and other diseases.

[0003] The formation of suspensions of microbubbles in an injectableliquid carrier suitable for echography can be produced by the release ofa gas dissolved under pressure in this liquid, or by a chemical reactiongenerating gaseous products, or by admixing with the liquid soluble orinsoluble solids containing air or gas trapped or adsorbed therein.

[0004] For instance, in U.S. Pat. No. 4,446,442 (Schering), there aredisclosed a series of different techniques for producing suspensions ofgas microbubbles in a sterilized injectable liquid carrier using (a) asolution of a tenside (surfactant) in a carrier liquid (aqueous) and (b)a solution of a viscosity enhancer as stabilizer. For generating thebubbles, the techniques disclosed there include forcing at high velocitya mixture of (a), (b) and air through a small aperture; or injecting (a)into (b) shortly before use together with a physiologically acceptablegas; or adding an acid to (a) and a carbonate to (b), both componentsbeing mixed together just before use and the acid reacting with thecarbonate to generate CO₂ bubbles; or adding an over-pressurized gas toa mixture of (a) and (b) under storage, said gas being released intomicrobubbles at the time when the mixture is used for injection.

[0005] EP-A-131,540 (Schering) discloses the preparation of microbubblesuspensions in which a stabilized injectable carrier liquid, e.g. aphysiological aqueous solution of salt, or a solution of a sugar likemaltose, dextrose, lactose or galactose, is mixed with solidmicroparticles (in the 0.1 to 1 μm range) of the same sugars containingentrapped air. In order to develop the suspension of bubbles in theliquid carrier, both liquid and solid components are agitated togetherunder sterile conditions for a few seconds and, once made, thesuspension must then be used immediately, i.e. it should be injectedwithin 5-10 minutes for echographic measurements; indeed, because theyare evanescent, the bubble concentration becomes too low for beingpractical after that period.

[0006] In an attempt to cure the evanescence problem, microballoons,i.e. microvesicles with a material wall, have been developed. As saidbefore, while the microbubbles only have an immaterial or evanescentenvelope, i.e. they are only surrounded by a wall of liquid whosesurface tension is being modified by the presence of a surfactant, themicroballoons or microcapsules have a tangible envelope made ofsubstantive material, e.g. a polymeric membrane with definite mechanicalstrength. In other terms, they are microvesicles of material in whichthe air or gas is more or less tightly encapsulated.

[0007] For instance, U.S. Pat. No. 4,276,885 (Tickner et al.) disclosesusing surface membrane microcapsules containing a gas for enhancingultrasonic images, the membrane including a multiplicity of non-toxicand non-antigenic organic molecules. In a disclosed embodiment, thesemicrobubbles have a gelatine membrane which resists coalescence andtheir preferred size is 5-10 μm. The membrane of these microbubbles issaid to be sufficiently stable for making echographic measurements.

[0008] Air-filled microballoons without gelatin are disclosed in U.S.Pat. No. 4,718,433 (Feinstein). These microvesicles are made bysonication (5 to 30 kHz) of protein solutions like 5% serum albumin andhave diameters in the 2-20 μm range, mainly 2-4 μm. The microvesiclesare stabilized by denaturation of the membrane forming protein aftersonication, for instance by using heat or by chemical means, e.g. byreaction with formaldehyde or glutaraldehyde. The concentration ofstable microvesicles obtained by this technique is said to be about8×10⁶/ml in the 2-4 μm range, about 10⁶/ml in the 4-5 μm range and lessthan 5× 10⁵ in the 5-6 μm range. The stability time of thesemicrovesicles is said to be 48 hrs or longer and they permit convenientleft heart imaging after intravenous injection. For instance, thesonicated albumin microbubbles when injected into a peripheral vein arecapable of transpulmonary passage. This results in echocardiographicopacification of the left ventricle cavity as well as myocardialtissues.

[0009] Recently, still further improved microballoons for injectionultrasonic echography have been reported in EP-A-324.938 (Widder). Inthis document there are disclosed high concentrations (more than 10⁸/ml)of air-filled protein-bounded microvesicles of less than 10 μm whichhave life-times of several months or more. Aqueous suspensions of thesemicroballoons are produced by ultrasonic cavitation of solutions of heatdenaturable proteins, e.g. human serum albumin, which operation alsoleads to a degree of foaming of the membrane-forming protein and itssubsequent hardening by heat. Other proteins such as hemoglobin andcollagen were also said to be convenient In this process. The highstorage stability of the suspensions of microballoons disclosed inEP-A-324.938 enables them to be marketed as such, i.e. with the liquidcarrier phase, which is a strong commercial asset since preparationbefore use is no longer necessary.

[0010] Similar advantages have been recently discovered in connectionwith the preparation of aqueous microbubble suspensions, i.e. there hasbeen discovered storage-stable dry pulverulent composition which willgenerate long-lasting bubble suspensions upon the addition of water.This is being disclosed in Application PCT/EP 91/00620 where liposomescomprising membrane-forming lipids are freeze-dried, and thefreeze-dried lipids, after exposure to air or a gas for a period oftime, will produce long-lasting bubble suspensions upon simple additionthereto of an aqueous liquid carrier.

[0011] Despite the many progresses achieved regarding the stabilityunder storage of aqueous microbubble suspensions, this being either inthe precursor or final preparation stage, there still remained until nowthe problem of vesicle durability when the suspensions are exposed tooverpressure, e.g. pressure variations such as that occurring afterinjection in the blood stream of a patient and consecutive to heartpulses, particularly in the left ventricle. Actually, the presentinventors have observed that, for instance in anaesthetised rabbits, thepressure variations are not sufficient to substantially alter the bubblecount for a period of time after injection. In contrast, in dogs andhuman patients, typical microbubbles or microballoons filled with commongases such as air, methane or CO₂ will collapse completely in a matterof seconds after injection due to the blood pressure effect. Thisobservation has been confirmed by others: For instance, S. GOTTLIEB etal. in J. Am. Soc. of Echocardiography 3 (1990) 238 have reported thatcross-linked albumin microballoons prepared by the sonication methodwere losing all echogenic properties after being subjected to anoverpressure of 60 Torr. It became hence important to solve the problemand to increase the useful life of suspensions of microbubbles andmembrane bounded microballoons under pressure in order to ensure thatechographic measurements can be performed in vivo safely andreproducibly.

[0012] It should be mentioned at this stage that another category ofechogenic image enhancing agents has been proposed which resistoverpressures as they consist of plain microspheres with a porousstructure, such porosity containing air or a gas. Such microspheres aredisclosed for instance in WO-A-91/12823 (DELTA BIOTECHNOLOGY), EP-A-327490 (SCHERING) and EP-A-458 079 (HOECHST. The drawback with the plainporous microspheres is that the encapsulated gas-filled free space isgenerally too small for good echogenic response and the spheres lackadequate elasticity. Hence the preference generally remains with thehollow microvesicles and a solution to the collapsing problem wassearched.

DISCLOSURE OF THE INVENTION

[0013] This problem has now been solved by using gases or gas mixturesin conformity with the criteria outlined in the claims. Briefly, it hasbeen found that when the echogenic microvesicles are made in thepresence of a gas, respectively are filled at least in part with a gas,having physical properties in conformity with the equation below, thenthe microvesicles remarkably resist pressure >60 Torr after injectionfor a time sufficient to obtain reproducible echographic measurements:${\frac{s_{gas}}{s_{air}} \times \frac{\sqrt{{Mw}_{air}}}{\sqrt{{Mw}_{gas}}}} \leq 1$

[0014] In the foregoing equation, “s” designates the solubilities inwater expressed as the “BUNSEN” coefficients, i.e. as volume of gasdissolved by unit volume of water under standard conditions (1 bar, 25°C.), and under partial pressure of the given gas of 1 atm (see the GasEncyclopaedia, Elsevier 1976). Since, under such conditions anddefinitions, the solubility of air is 0.0167, and the square root of itsaverage molecular weight (Mw) is 5.39, the above relation simplifies to:

s_(gas)/{square root}Mw_(gas)≦0.0031

[0015] In the Examples to be found hereafter there is disclosed thetesting of echogenic microbubbles and microballoons (see the Tables)filled with a number of different gases and mixtures thereof, and thecorresponding resistance thereof to pressure increases, both in vivo andin vitro. In the Tables, the water solubility factors have also beentaken from the aforecited Gas Encyclopaedia from “L'Air Liquide”.Elsevier Publisher (1976).

[0016] The microvesicles in aqueous suspension containing gasesaccording to the invention include most microbubbles and microballoonsdisclosed until now for use as contrast agents for echography. Thepreferred microballoons are those disclosed in EP-A-324.938,PCT/EP91/01706 and EP-A-458 745; the preferred microbubbles are those ofPCT/EP91/00620; these microbubbles are advantageously formed from anaqueous liquid and a dry powder (microvesicle precursors) containinglamellarized freeze-dried phospholipids and stabilizers; themicrobubbles are developed by agitation of this powder in admixture withthe aqueous liquid carrier. The microballoons of EP-A-458 745 have aresilient interfacially precipitated polymer membrane of controlledporosity. They are generally obtained from emulsions into microdropletsof polymer solutions in aqueous liquids, the polymer being subsequentlycaused to precipitate from its solution to form a filmogenic membrane atthe droplet/liquid interface, which process leads to the initialformation of liquid-filled microvesicles, the liquid core thereof beingeventually substituted by a gas.

[0017] In order to carry out the method of the present invention, i.e.to form or fill the microvesicles, whose suspensions in aqueous carriersconstitute the desired echogenic additives, with the gases according tothe foregoing relation, one can either use, as a first embodiment, a twostep route consisting of (1) making the microvesicles from appropriatestarting materials by any suitable conventional technique in thepresence of any suitable gas, and (2) replacing this gas originally used(first gas) for preparing the microvesicles with a new gas (second gas)according to the invention (gas exchange technique).

[0018] Otherwise, according to a second embodiment, one can directlyprepare the desired suspensions by suitable usual methods under anatmosphere of the new gas according to the invention.

[0019] If one uses the two-step route, the initial gas can be firstremoved from the vesicles (for instance by evacuation under suction) andthereafter replaced by bringing the second gas into contact with theevacuated product, or alternatively, the vesicles still containing thefirst gas can be contacted with the second gas under conditions wherethe second gas will displace the first gas from the vesicles (gassubstitution). For instance, the vesicle suspensions, or preferablyprecursors thereof (precursors here may mean the materials themicrovesicle envelopes are made of, or the materials which, uponagitation with an aqueous carrier liquid, will generate or develop theformation of microbubbles in this liquid), can be exposed to reducedpressure to evacuate the gas to be removed and then the ambient pressureis restored with the desired gas for substitution. This step can berepeated once or more times to ensure complete replacement of theoriginal gas by the new one. This embodiment applies particularly wellto precursor preparations stored dry, e.g. dry powders which willregenerate or develop the bubbles of the echogenic additive uponadmixing with an amount of carrier liquid. Hence, in one preferred casewhere microbubbles are to be formed from an aqueous phase and drylaminarized phospholipids, e.g. powders of dehydrated lyophilizedliposomes plus stabilizers, which powders are to be subsequentlydispersed under agitation in a liquid aqueous carrier phase, it isadvantageous to store this dry powder under an atmosphere of a gasselected according to the invention. A preparation of such kind willkeep indefinitely in this state and can be used at any time fordiagnosis, provided it is dispersed into sterile water before injection.

[0020] Otherwise, and this is particularly so when the gas exchange isapplied to a suspension of microvesicles in a liquid carrier phase, thelatter is flushed with the second gas until the replacement (partial orcomplete) is sufficient for the desired purpose. Flushing can beeffected by bubbling from a gas pipe or, in some cases, by simplysweeping the surface of the liquid containing the vesicles under gentleagitation with a stream (continuous or discontinuous) of the new gas. Inthis case, the replacement gas can be added only once in the flaskcontaining the suspension and allowed to stand as such for a while, orit can be renewed one or more times in order to assure that the degreeof renewal (gas exchange) is more or less complete.

[0021] Alternatively, in a second embodiment as said before, one willeffect the full preparation of the suspension of the echogenic additivesstarting with the usual precursors thereof (starting materials), asrecited in the prior art and operating according to usual means of saidprior art, but in the presence of the desired gases or mixture of gasesaccording to the invention instead of that of the prior art whichusually recites gases such as air, nitrogen, CO₂ and the like.

[0022] It should be noted that in general the preparation mode involvingone first type of gas for preparing the microvesicles and, thereafter,substituting the original gas by a second kind of gas, the latter beingintended to confer different echogenic properties to said microvesicles,has the following advantage: As will be best seen from the results inthe Examples hereinafter, the nature of the gas used for making themicrovesicles, particularly the microballoons with a polymer envelope,has a definitive influence on the overall size (i.e. the average meandiameter) of said microvesicles; for instance, the size of microballoonsprepared under air with precisely set conditions can be accuratelycontrolled to fall within a desired range, e.g. the 1 to 10 μm rangesuitable for echographying the left and right heart ventricles. This notso easy with other gases, particularly the gases in conformity with therequirements of the present invention; hence, when one wishes to obtainmicrovesicles in a given size range but filled with gases the nature ofwhich would render the direct preparation impossible or very hard, onewill much advantageously rely on the two-steps preparation route, i.e.one will first prepare the microvesicles with a gas allowing moreaccurate diameter and count control, and thereafter replace the firstgas by a second gas by gas exchange.

[0023] In the description of the Experimental part that follows(Examples), gas-filled microvesicles suspended in water or other aqueoussolutions have been subjected to pressures over that of ambient. It wasnoted that when the overpressure reached a certain value (which isgenerally typical for a set of microsphere parameters and workingconditions like temperature, compression rate, nature of carrier liquidand its content of dissolved gas (the relative importance of thisparameter will be detailed hereinafter), nature of gas filler, type ofechogenic material, etc.), the microvesicles started to collapse, thebubble count progressively decreasing with further increasing thepressure until a complete disappearance of the sound reflector effectoccurred. This phenomenon was better followed optically, (nephelometricmeasurements) since it is paralleled by a corresponding change inoptical density, i.e. the transparency of the medium increases as thebubble progressively collapse. For this, the aqueous suspension ofmicrovesicles (or an appropriate dilution thereof was placed in aspectrophotometric cell maintained at 25° C. (standard conditions) andthe absorbance was measured continuously at 600 or 700 nm, while apositive hydrostatic overpressure was applied and gradually increased.The pressure was generated by means of a peristaltic pump (GILSON'sMini-puls) feeding a variable height liquid column connected to thespectrophotometric cell, the latter being sealed leak-proof. Thepressure was measured with a mercury manometer calibrated in Torr. Thecompression rate with time was found to be linearly correlated with thepump's speed (rpm's). The absorbance in the foregoing range was found tobe proportional to the microvesicle concentration in the carrier liquid.

[0024]FIG. 1 is a graph which relates the bubble concentration (bubblecount), expressed in terms of optical density in the aforementionedrange, and the pressure applied over the bubble suspension. The data forpreparing the graph are taken from the experiments reported in Example4.

[0025]FIG. 1 shows graphically that the change of absorbance versuspressure is represented by a sigmoid-shaped curve. Up to a certainpressure value, the curve is nearly flat which indicates that thebubbles are stable. Then, a relatively fast absorbance drop occurs,which indicates the existence of a relatively narrow critical regionwithin which any pressure increase has a rather dramatic effect on thebubble count. When all the microvesicles have disappeared, the curvelevels off again. A critical point on this curve was selected in themiddle between the higher and lower optical readings, i.e. intermediatebetween the “full”-bubble (OD max) and the “no”-bubble (OD min)measurements, this actually corresponding where about 50% of the bubblesinitially present have disappeared, i.e. where the optical densityreading is about half the initial reading, this being set, in the graph,relative to the height at which the transparency of the pressurizedsuspension is maximal (base line). This point which is also in thevicinity where the slope of the curve is maximal is defined as thecritical pressure PC. It was found that for a given gas, PC does notonly depend on the aforementioned parameters but also, and particularlyso, on the actual concentration of gas (or gases) already dissolved inthe carrier liquid: the higher the gas concentration, the higher thecritical pressure. In this connection, one can therefore increase theresistance to collapse under pressure of the microvesicles by making thecarrier phase saturated with a soluble gas, the latter being the same,or not, (i.e. a different gas) as the one that fills the vesicles. As anexample, air-filled microvesicles could be made very resistant tooverpressures (>120 Torr) by using, as a carrier liquid, a saturatedsolution of CO₂. Unfortunately, this finding is of limited value in thediagnostic field since once the contrast agent is injected to thebloodstream of patients (the gas content of which is of course outsidecontrol), it becomes diluted therein to such an extent that the effectof the gas originally dissolved in the injected sample becomesnegligible.

[0026] Another readily accessible parameter to reproducibly compare theperformance of various gases as microsphere fillers is the width of thepressure interval (ΔP) limited by the pressure values under which thebubble counts (as expressed by the optical densities) is equal to the75% and 25% of the original bubble count. Now, it has been surprisinglyfound that for gases where the pressure difference DP=P₂₅-P₇₅ exceeds avalue of about 25-30 Torr, the killing effect of the blood pressure onthe gas-filled microvesicles is minimized, i.e. the actual decrease inthe bubble count is sufficiently slow not to impair the significance,accuracy and reproducibility of echographic measurements.

[0027] It was found, in addition, that the values of PC and ΔP alsodepend on the rate of rising the pressure in the test experimentsillustrated by FIG. 1, i.e. in a certain interval of pressure increaserates (e.g. in the range of several tens to several hundreds ofTorr/min), the higher the rate, the larger the values for PC and ΔP. Forthis reason, the comparisons effected under standard temperatureconditions were also carried out at the constant increase rate of 100Torr/min. It should however be noted that this effect of the pressureincrease rate on the measure of the PC and ΔP values levels off for veryhigh rates: for instance the values measured under rates of severalhundreds of Torr/min are not significantly different from those measuredunder conditions ruled by heart beats.

[0028] Although the very reasons why certain gases obey theaforementioned properties, while others do not, have not been entirelyclarified, it would appear that some relation possibly exists in which,in addition to molecular weight and water solubility, dissolutionkinetics, and perhaps other parameters, are involved. However theseparameters need not be known to practise the present invention since gaseligibility can be easily determined according to the aforediscussedcriteria.

[0029] The gaseous species which particularly suit the invention are,for instance, halogenated hydrocarbons like the freons and stablefluorinated chalcogenides like SF₆, SeF₆ and the like.

[0030] It has been mentioned above that the degree of gas saturation ofthe liquid used as carrier for the microvesicles according to theinvention has an importance on the vesicle stability under pressurevariations. Indeed, when the carrier liquid in which the microvesiclesare dispersed for making the echogenic suspensions of the invention issaturated at equilibrium with a gas, preferably the same gas with whichthe microvesicles are filled, the resistance of the microvesicles tocollapse under variations of pressure is markedly increased. Thus, whenthe product to be used as a contrast agent is sold dry to be mixed justbefore use with the carrier liquid (see for instance the productsdisclosed in PCT/EP91/00620 mentioned hereinbefore). it is quiteadvantageous to use, for the dispersion, a gas saturated aqueouscarrier. Alternatively, when marketing ready-to-use microvesiclesuspensions as contrast agents for echography, one will advantageouslyuse as the carrier liquid for the preparation a gas saturated aqueoussolution; in this case the storage life of the suspension will beconsiderably increased and the product may be kept substantiallyunchanged (no substantial bubble count variation) for extended periods,for instance several weeks to several months, and even over a year inspecial cases. Saturation of the liquid with a gas may be effected mosteasily by simply bubbling the gas into the liquid for a period of timeat room temperature.

EXAMPLE 1

[0031] Albumin microvesicles filled with air or various gases wereprepared as described in EP-A- 324 938 using a 10 ml calibrated syringefilled with a 5% human serum albumin (HSA) obtained from the BloodTransfusion Service, Red-Cross Organization, Bern, Switzerland. Asonicator probe (Sonifier Model 250 from Branson Ultrasonic Corp. USA)was lowered into the solution down to the 4 ml mark of the syringe andsonication was effected for 25 sec (energy setting=8). Then thesonicator probe was raised above the solution level up to the 6 ml markand sonication was resumed under the pulse mode (cycle= 0.3) for 40 sec.After standing overnight at 4° C., a top layer containing most of themicrovesicles had formed by buoyancy and the bottom layer containingunused albumin debris of denatured protein and other insolubles wasdiscarded. After resuspending the microvesicles in fresh albuminsolution the mixture was allowed to settle again at room temperature andthe upper layer was finally collected. When the foregoing sequences werecarried out under the ambient atmosphere, air filled microballoons wereobtained. For obtaining microballoons filled with other gases, thealbumin solution was first purged with a new gas, then the foregoingoperational sequences were effected under a stream of this gas flowingon the surface of the solution; then at the end of the operations, thesuspension was placed in a glass bottle which was extensively purgedwith the desired gas before sealing.

[0032] The various suspensions of microballoons filled with differentgases were diluted to 1:10 with distilled water saturated at equilibriumwith air, then they were placed in an optical cell as described aboveand the absorbance was recorded while increasing steadily the pressureover the suspension. During the measurements, the suspensionstemperature was kept at 25° C.

[0033] The results are shown in the Table 1 below and are expressed interms of the critical pressure PC values registered for a series ofgases defined by names or formulae, the characteristic parameters ofsuch gases, i.e. Mw and water solubility being given, as well as theoriginal bubble count and bubble average size (mean diameter in volume).TABLE 1 Bubble Bubble count size PC S_(gas) Sample Gas Mw Solubility(10⁸/ml) (μm) (Torr) {square root}Mw AFre1 CF₄ 88 .0038 0.8 5.1 120.0004 AFre2 CBrF₃ 149 .0045 0.1 11.1 104 .0004 ASF1 SF₆ 146 .005 13.96.2 150 .0004 ASF2 SF₆ 146 .005 2.0 7.9 140 .0004 AN1 N₂ 28 .0144 0.47.8 62 .0027 A14 Air 29 .0167 3.1 11.9 53 .0031 A18 Air 29 .0167 3.8 9.252 — A19 Air 29 .0167 1.9 9.5 51 — AMe1 CH₄ 16 .032 0.25 8.2 34 .008 AKr1 Kr 84 .059 0.02 9.2 86 .006  AX1 Xe 131 .108 0.06 17.2 65 .009  AX2Xe 131 .108 0.03 16.5 89 .009 

[0034] From the results of Table 1, it is seen that the criticalpressure PC increases for gases of lower solubility and higher molecularweight. It can therefore be expected that microvesicles filled with suchgases will provide more durable echogenic signals in vivo. It can alsobe seen that average bubble size generally increases with gassolubility.

EXAMPLE 2

[0035] Aliquots (1 ml) of some of the microballoon suspensions preparedin Example 1 were injected in the jugular vein of experimental rabbitsin order to test echogenicity in vivo. Imaging of the left and rightheart ventricles was carried out in the grey scale mode using an Acuson128-XP5 echography apparatus and a 7.5 MHz transducer. The duration ofcontrast enhancement in the left ventricle was determined by recordingthe signal for a period of time. The results are gathered in Table 2below which also shows the PC of the gases used. TABLE 2 Duration ofSample (Gas) contrast (sec) PC (Torr) AMe1 (CH₄) zero 34 A14 (air) 10 53A18 (air) 11 52 AX1 (Xe) 20 65 AX2 (Xe) 30 89 ASF2 (SF₆) >60   140 

[0036] From the above results, one can see the existence of a definitecorrelation between the critical pressure of the gases tried and thepersistence in time of the echogenic signal.

EXAMPLE 3

[0037] A suspension of echogenic air-filled galactose microparticles(Echovist® from SCHERING AG) was obtained by shaking for 5 sec 3 g ofthe solid microparticles in 8.5 ml of a 20% galactose solution. In otherpreparations, the air above a portion of Echovist® particles wasevacuated (0.2 Torr) and replaced by an SF₆ atmosphere, whereby, afteraddition of the 20% galactose solution, a suspension of microparticlescontaining associated sulfur hexafluoride was obtained. Aliquots (1 ml)of the suspensions were administered to experimental rabbits (byinjection in the jugular vein) and imaging of the heart was effected asdescribed in the previous example. In this case the echogenicmicroparticles do not transit through the lung capillaries, henceimaging is restricted to the right ventricle and the overall signalpersistence has no particular significance. The results of Table 3 belowshow the value of signal peak intensity a few seconds after injection.TABLE 3 Signal peak Sample No Gas (arbitrary units) Gal1 air 114 Gal2air 108 Gal3 SF₆ 131 Gal4 SF₆ 140

[0038] It can be seen that sulfur hexafluoride, an inert gas with lowwater solubility, provides echogenic suspensions which generateechogenic signals stronger than comparable suspensions filled with air.These results are particularly interesting in view of the teachings ofEP-A-441 468 and 357 163 (SCHERING) which disclose the use forechography purposes of micropartcles, respectively, cavitate andclathrate compounds filled with various gases including SF6; thesedocuments do not however report particular advantages of SF6 over othermore common gases with regard to the echogenic response.

EXAMPLE 4

[0039] A series of echogenic suspensions of gas-filled microbubbles wereprepared by the general method set forth below:

[0040] One gram of a mixture of hydrogenated soya lecithin (fromNattermann Phospholipids GmbH, Germany) and dicetyl-phosphate (DCP), in9/1 molar ratio, was dissolved in 50 ml of chloroform, and the solutionwas placed in a 100 ml round flask and evaporated to dryness on aRotavapor apparatus. Then, 20 ml of distilled water were added and themixture was slowly agitated at 75° C. for an hour. This resulted in theformation of a suspension of multilamellar liposomes (MLV) which wasthereafter extruded at 75° C. through, successively, 3 μm and 0.8 μmpolycarbonate membranes (Nuclepore®). After cooling, 1 ml aliquots ofthe extruded suspension were diluted with 9 ml of a concentrated lactosesolution (83 g/l), and the diluted suspensions were frozen at −45° C.The frozen samples were thereafter freeze-dried under high vacuum to afree-flowing powder in a vessel which was ultimately filled with air ora gas taken from a selection of gases as indicated in Table 4 below. Thepowdery samples were then resuspended in 10 ml of water as the carrierliquid, this being effected under a stream of the same gas used to fillthe said vessels. Suspension was effected by vigorously shaking for 1min on a vortex mixer.

[0041] The various suspensions were diluted 1:20 with distilled waterequilibrated beforehand with air at 25° C. and the dilutions were thenpressure tested at 25° C. as disclosed in Example 1 by measuring theoptical density in a spectrophotometric cell which was subjected to aprogressively increasing hydrostatic pressure until all bubbles hadcollapsed. The results are collected in Table 4 below which, in additionto the critical pressure PC, gives also the ΔP values (see FIG. 1).TABLE 4 Bubble Sample Solubility count PC ΔP No Gas M_(w) in H₂O(10⁸/ml) (Torr) (Torr) LFre1 CF₄ 88 .0038 1.2 97 35 LFre2 CBrF₃ 149.0045 0.9 116  64 LSF1 SF₆ 146 .005 1.2 92 58 LFre3 C₄F₈ 200 .016 1.5136  145  L1 air 29 .0167 15.5 68 17 L2 air 29 .0167 11.2 63 17 LAr1 Ar40 .031 14.5 71 18 LKr1 Kr 84 .059 12.2 86 18 LXe1 Xe 131 .108 10.1 9223 LFre4 CHClF₂ 86 .78 — 83 25

[0042] The foregoing results clearly indicate that the highestresistance to pressure increases is provided by the most water-insolublegases. The behavior of the microbubbles is therefore similar to that ofthe microballoons in this regard. Also, the less water-soluble gaseswith the higher molecular weights provide the flattestbubble-collapse/pressure curves (i.e. ΔP is the widest) which is also animportant factor of echogenic response durability in vivo, as indicatedhereinbefore.

EXAMPLE 5

[0043] Some of the microbubble suspensions of Example 4 were injected tothe jugular vein of experimental rabbits as indicated in Example 2 andimaging of the left heart ventricle was effected as indicatedpreviously. The duration of the period for which a useful echogenicsignal was detected was recorded and the results are shown in Table 5below in which C₄F₈ indicates octafluorocyclobutane. TABLE 5 Contrastduration Sample No Type of gas (sec) L1 Air 38 L2 Air 29 LMe1 CH₄ 47LKr1 Krypton 37 LFre1 CF₄ >120 LFre2 CBrF₃ 92 LSF1 SF₆ >112 LFre3 C₄F₈>120

[0044] These results indicate that, again in the case of microbubbles,the gases according to the criteria of the present invention willprovide ultrasonic echo signal for a much longer period than most gasesused until now.

EXAMPLE 6

[0045] Suspensions of microbubbles were prepared using different gasesexactly as described in Example 4, but replacing the lecithinphospholipid ingredient by a mole equivalent ofdiarachidoyl-phosphatidylcholine (C₂₀ fatty acid residue) available fromAvanti Polar Lipids, Birmingham, Ala., USA. The phospholipid to DCPmolar ratio was still 9/1. Then the suspensions were pressure tested asin Example 4; the results, collected in Table 6A below, are to becompared with those of Table 4. TABLE 6A Solubi- Bubble Sample Type ofMw of lity in count PC ΔP No gas gas water (10⁸/ml) (Torr) (Torr) LFre1CF₄ 88 .0038 3.4 251 124 LFre2 CBrF₃ 149 .0045 0.7 121 74 LSF1 SF₆ 146.005 3.1 347 >150 LFre3 C₄F₈ 200 .016 1.7 >350 >200 L1 Air 29 .0167 3.860 22 LBu1 Butane 58 .027 0.4 64 26 LAr1 Argon 40 .031 3.3 84 47 LMe1CH₄ 16 .032 3.0 51 19 LEt1 C₂H₆ 44 .034 1.4 61 26 LKr1 Kr 84 .059 2.7 6318 LXe1 Xe 131 .108 1.4 60 28 LFre4 CHClF₂ 86 .78 0.4 58 28

[0046] The above results, compared to that of Table 4, show that, atleast with low solubility gases, by lengthening the chain of thephospholipid fatty acid residues, one can dramatically increase thestability of the echogenic suspension toward pressure increases. Thiswas further confirmed by repeating the foregoing experiments butreplacing the phospholipid component by its higher homolog, i.e.di-behenoyl-phosphatidylcholine (C₂₂ fatty acid residue). In this case,the resistance to collapse with pressure of the microbubbles suspensionswas still further increased.

[0047] Some of the microbubbles suspensions of this Example were testedin dogs as described previously for rabbits (imaging of the heartventricles after injection of 5 ml samples in the anterior cephalicvein). A significant enhancement of the useful in-vivo echogenicresponse was noted, in comparison with the behavior of the preparationsdisclosed in Example 4, i.e. the increase in chain length of thefatty-acid residue in the phospholipid component increases the usefullife of the echogenic agent in-vivo.

[0048] In the next Table below, there is shown the relative stability inthe left ventricle of the rabbit of microbubbles (SF₆) prepared fromsuspensions of a series of phospholipids whose fatty acid residues havedifferent chain lengths (<injected dose: 1 ml/rabbit). TABLE 6B Chainlength PC ΔP Duration of Phospholipid (C_(n)) (Torr) (Torr) contrast(sec) DMPC 14  57 37  31 DPPC 16 100 76 105 DSPC 18 115 95 120 DAPC 20266 190  >300 

[0049] It has been mentioned hereinabove that for the measurement ofresistance to pressure described in these Examples, a constant rate ofpressure rise of 100 Torr/min was maintained. This is justified by theresults given below which show the variations of the PC values fordifferent gases in function to the rate of pressure increase. In thesesamples DMPC was the phospholipid used. PC (Torr) Gas Rate of pressureincrease (Torr/min) sample 40 100 200 SF₆ 51 57 82 Air 39 50 62 CH₄ 4761 69 Xe 38 43 51 Freon 22 37 54 67

EXAMPLE 7

[0050] A series of albumin microballoons as suspensions in water wereprepared under air in a controlled sphere size fashion using thedirections given in Example 1. Then the air in some of the samples wasreplaced by other gases by the gas-exchange sweep method at ambientpressure. Then, after diluting to 1:10 with distilled water as usual,the samples were subjected to pressure testing as in Example 1. From theresults gathered in Table 7 below, it can be seen that the two-stepspreparation mode gives, in some cases, echo-generating agents withbetter resistance to pressure than the one-step preparation mode ofExample 1. TABLE 7 Initial bubble Sample Type of Mw of the Solubilitycount PC No gas gas in water (10⁸/ml) (Torr) A14 Air 29 .0167 3.1 53 A18Air 29 .0167 3.8 52 A18/SF₆ SF₆ 146 .005 0.8 115 A18/C₂H₆ C₂H₆ 30 .0423.4 72 A19 Air 29 .0167 1.9 51 A19/SF₆ SF₆ 146 .005 0.6 140 A19/Xe Xe131 .108 1.3 67 A22/CF₄ CF₄ 88 .0038 1.0 167 A22/Kr Kr 84 .059 0.6 85

EXAMPLE 8

[0051] The method of the present invention was applied to an experimentas disclosed in the prior art, for instance Example 1 WO-92/11873. Threegrams of Pluronic® F68 (a copolymer of polyoxyethylene-polyoxypropylenewith a molecular weight of 8400), 1 g of dipalmitoylphosphatidylglycerol(Na salt, AVANTI Polar Lipids) and 3.6 g of glycerol were added to 80 mlof distilled water. After heating at about 80° C., a clear homogenoussolution was obtained. The tenside solution was cooled to roomtemperature and the volume was adjusted to 100 ml. In some experiments(see Table 8) dipalmitoylphosphatidyl-glycerol was replaced by a mixtureof diarachidoylphosphatidylcholine (920 mg) and 80 mg ofdipalmitoylphosphatidic acid (Na salt, AVANTI Polar lipids).

[0052] The bubble suspensions were obtained by using two syringesconnected via a three-way valve. One of the syringes was filled with 5ml of the tenside solution while the other was filled with 0.5 ml of airor gas. The three-way valve was filled with the tenside solution beforeit was connected to the gas-containing syringe. By alternativelyoperating the two pistons, the tenside solutions were transferred backand forth between the two syringes (5 times in each direction), milkysuspensions were formed. After dilution (1:10 to 1:50) with distilledwater saturated at equilibrium with air, the resistance to pressure ofthe preparations was determined according to Example 1. The pressureincrease rate was 240 Torr/min. The following results were obtained:TABLE 8 Phospholipid Gas Pc (mm Hg) DP (mm Hg) DPPG air  28  17 DPPG SF₆138 134 DAPC/DPPA 9/1 air  46  30 DAPC/DPPA 9/1 SF₆ 269 253

[0053] It follows that by using the method of the invention andreplacing air with other gases e.g. SF₆ even with known preparations aconsiderable improvements i.e. increase in the resistance to pressuremay be achieved. This is true both in the case of negatively chargedphospholipids (e.g. DPPG) and in the case of mixtures of neutral andnegatively charged phospholipids (DAPC/DPPA).

[0054] The above experiment further demonstrates that the recognisedproblem sensitivity of microbubbles and microballoons to collapse whenexposed to pressure i.e. when suspensions are injected into livingbeings, has advantageously been solved by the method of the invention.Suspensions with microbubbles or microballoons with greater resistanceagainst collapse and greater stability can advantageously be producedproviding suspensions with better reproducibility and improved safety ofechographic measurements performed in vivo on a human or animal body.

What is claimed is:
 1. A method for imparting resistance againstcollapsing to contrast agents for ultrasonic echography which consist ofgas-filled microvesicles in suspension in aqueous liquid carrier phases,i.e. either microbubbles bounded by an evanescent gas/liquid interfacialclosed surface, or microballoons bounded by a material envelope, saidcollapsing resulting, at least in part, from pressure increaseseffective e.g. when the said suspensions are injected into thebloodstream of patients, said method comprising forming saidmicrovesicles in the presence of a gas, or if the microvesicles arealready made filling them with this gas, which is a physiologicallyacceptable gas, or gas mixture, at least a fraction of which has asolubility in water expressed in liters of gas by liter of water understandard conditions divided by the square root of the molecular weightin daltons which does not exceed 0.003.
 2. The method of claim 1 , whichis carried out in two steps, in the first step the microvesicles or dryprecursors thereof are initially prepared under an atmosphere of a firstgas, then in the second step at least a fraction of the first gas issubstantially substituted by a second gas, the latter being saidphysiologically acceptable gas.
 3. The method of claim 1 , in which thephysiologically acceptable gas used is selected from SF₆, SeF₆, Freon®such as CF₄, CBrF₃, C₄F₈, CClF₃, CCl₂F₂, C₂F₆, C₂ClF₅, CBrClF₂, C₂Cl₂F₄,CBr₂F₂ and C₄F₁₀.
 4. The method of claim 2 , in which the gas used inthe first step is of a kind that allows effective control of the averagesize and concentration of the microvesicles in the carrier liquid, andthe physiologically acceptable gas added in the second step ensuresprolonged useful echogenic life to the suspension for in-vivo ultrasonicimaging.
 5. The method of claim 1 , in which the aqueous phase carryingthe microbubbles contains dissolved film-forming surfactants in lamellaror laminar form, said surfactants stabilizing the microbubbles boundaryat the gas to liquid interface.
 6. The method of claim 5 , in which saidsurfactants comprise one or more phospholipids.
 7. The method of claim 6, in which at least part of the phospholipids are in the form ofliposomes.
 8. The method of claim 6 , in which at least one of thephospholipids is a diacylphosphatidyl compound wherein the acyl group isa C₁₆ fatty acid residue or a higher homologue thereof.
 9. The method ofclaims 1 and 2, in which the microballoon material envelope is made ofan organic polymeric membrane.
 10. The method of claim 9 , in which thepolymers of the membrane are selected from polylactic or polyglycolicacid and their copolymers, reticulated serum albumin, reticulatedhaemoglobin, polystyrene, and esters of polyglutamic and polyasparticacids.
 11. The method of claim 1 , in which the forming of themicrovesicles with said physiologically acceptable gas is effected byalternately subjecting dry precursors thereof to reduced pressure andrestoring the pressure with said gas, and finally dispersing theprecursors in a liquid carrier.
 12. The method of claim 1 , in which thefilling of the microballoons with said physiologically acceptable gas iseffected by simply flushing the suspension with said gas under ambientpressure.
 13. The method of claim 1 , which comprises making themicrovesicles by any standard method known in the art but operatingunder an atmosphere composed at least in part of said gas. 14.Suspensions of gas filled microvesicles distributed in an aqueouscarrier liquid to be used as contrast agents in ultrasonic echography,characterized in that the gas is physiologically acceptable and suchthat at least a portion thereof has a solubility in water, expressed inliter of gas by liter of water under standard conditions, divided by thesquare root of the molecular weight which does not exceed 0.003.
 15. Theaqueous suspensions of claim 14 , characterized in that the gas is suchthat the pressure difference ΔP between those pressures which, whenapplied under standard conditions and at a rate of about 100 Torr/min tothe suspension cause the collapsing of about 75%, respectively 25%, ofthe microvesicles initially present, is at least 25 Torr.
 16. Aqueoussuspensions according to claim 14 , in which the microvesicles aremicrobubbles filled with said physiologically acceptable gas suspendedin an aqueous carrier liquid containing phospholipids whose fatty acidresidues contain 16 carbons or more.
 17. Contrast agents for echographyin precursor form consisting of a dry powder comprising lyophilizedliposomes and stabilizers, this powder being dispersible in aqueousliquid carriers to form echogenic suspensions of gas-filledmicrobubbles, characterized in that it is stored under an atmospherecomprising a physiologically acceptable gas whose solubility in water,expressed in liter of gas by liter of water under standard conditions,divided by the square root of the molecular weight does not exceed0.003.
 18. The contrast agent precursors of claim 17 , in which theliposomes comprise phospholipids whose fatty acid residues have 16 ormore carbon atoms.